2 Regulation of lipid metabolism

A high-fat diet causes metabolic disorders by disturbing lipogenesis and lipolysis, often leading to NAFLD. AMP-activated kinase (AMPK) is a key energy sensor that ameliorates NAFLD by increasing fatty acid utilisation and inhibiting hepatic lipid synthesis^{(Reference Woods, Williams, Muckett, Mayer, Liljevald, Bohlooly-Y and Carling20–Reference Garcia, Hellberg, Chaix, Wallace, Herzig, Badur, Lin, Shokhirev, Pinto, Ross, Saghatelian, Panda, Dow, Metallo and Shaw22)}. Inhibition of AMPK increases the risk of obesity and diabetes as well as NAFLD^{(Reference Weikel, Ruderman and Cacicedo23,Reference Dahlhoff, Worsch, Sailer, Hummel, Fiamoncini, Uebel, Obeid, Scherling, Geisel, Bader and Daniel24)} . High-fat diet-induced obesity-mediated NAFLD results from increased liver NEFA concentrations and triacylglycerol accumulation accompanied by inhibition of betaine homocysteine methyltransferase (BHMT) and decrease in S-adenosylmethionine (SAM) levels. Both BHMT and SAM down-regulate de novo lipogenesis by enhancing the expression of AMPK; therefore, methyl donor supplementation prevents the progression of hepatic steatosis associated with decreased hepatic triacylglycerol accumulation by increasing the activity of AMPK in obesity-mediated NAFLD mice^{(Reference Dahlhoff, Worsch, Sailer, Hummel, Fiamoncini, Uebel, Obeid, Scherling, Geisel, Bader and Daniel24)}. One study also reported that a high-fat diet disturbs the homeostasis of hepatic methionine metabolism, leading to fatty liver; betaine, as a methyl donor, prevents fatty liver and hepatic injury by increasing SAM levels and preventing changes in BHMT mRNA expression in rats^{(Reference Deminice, Silva, Lamarre, Kelly, Jacobs, Brosnan and Brosnan25)}. Additionally, betaine stimulates energy production and fatty acid synthesis by phosphorylation of AMPK in C2C12 myotubes^{(Reference Ma, Meng, Kang, Zhang, Jung and Park26)}. These studies indicate that betaine supplementation elevates SAM and BHMT levels via one-carbon metabolites and activates AMPK expression to reduce hepatic NEFAs, thus ameliorating NAFLD.

Although fatty acid and cholesterol synthesis is dependent on acetyl-CoA, the biosynthetic pathways are regulated by distinct sterol regulatory element binding proteins (SREBP). AMPK is the upstream protein of SREBP-1c^{(Reference Javary, Allain-Courtois, Saucisse, Costet, Heraud, Benhamed, Pierre, Bure, Pallares-Lupon, Do Cruzeiro, Postic, Cota, Dubus, Rosenbaum and Benhamouche-Trouillet27)} and carbohydrate response element-binding protein (ChREBP)^{(Reference Song, Deaciuc, Zhou, Song, Chen, Hill and McClain17)}. Both SREBP-1c and ChREBP are key transcription factors in the process of lipogenesis. Betaine decreases fatty acid synthesis and protects against hepatic steatosis by inhibiting the expression of SREBP-1c^{(Reference Ahn, Choi, Hong, Jun, Na, Choi and Kim28)}, mainly by activating AMPK, which also inhibits fatty acid synthase (FASN), stearoyl-CoA desaturase (SCD)^{(Reference Chen, Liu, Yu, Li, Wang, Zhang, Qiu and Wang21)}, ChREBP^{(Reference Song, Deaciuc, Zhou, Song, Chen, Hill and McClain17)} and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) expression in the liver^{(Reference Loh, Tam, Murray-Segal, Huynh, Meikle, Scott, van Denderen, Chen, Steel, LeBlond, Burkovsky, O’Dwyer, Nunes, Steinberg, Fullerton, Galic and Kemp29)}. Acetyl-CoA carboxylase (ACC) catalyses acetyl-CoA conversion to malonyl-CoA and is the first committed step in de novo lipogenesis. Therefore, the inhibitory effects of betaine on fatty acid synthesis are mediated by phosphorylation of AMPK^{(Reference Ma, Meng, Kang, Zhang, Jung and Park26)}, which phosphorylates ACC^{(Reference Esquejo, Salatto, Delmore, Albuquerque, Reyes, Shi, Moccia, Cokorinos, Peloquin, Monetti, Barricklow, Bollinger, Smith, Day, Nguyen, Geoghegan, Kreeger, Opsahl, Ward, Kalgutkar, Tess, Butler, Shirai, Osborne, Steinberg, Birnbaum, Cameron and Miller30)}, leading to a decrease in the production of malonyl-CoA. Malonyl-CoA is converted to mevalonate by reduction to mevalonic acid by HMGCR, a key rate-limiting enzyme in cholesterol biosynthesis. Betaine also inhibits HMGCR to decrease the synthesis of cholesterol via AMPK phosphorylation^{(Reference Ma, Meng, Kang, Zhang, Jung and Park26)}.

Hepatic steatosis in high-fructose diet-induced NAFLD rats is related to overexpression of liver X receptor (LXR)^{(Reference Maithilikarpagaselvi, Sridhar, Swaminathan, Sripradha and Badhe31)}, which not only significantly increases intracellular triacylglycerol content by increasing SREBP-1c, FAS and SCD1 expression in HepG2 cells^{(Reference Sim, Kim, Lee, Choi, Choi, Shin, Jun, Park, Park, Kim, Oh and Lee32)} but also increases cholesterol levels by up-regulating the expression of CYP7A1^{(Reference Cai, Yuan, Liu, Pan, Ma, Hong and Zhao33)}. Betaine plays a protective role in decreasing triacylglycerol mainly by depressing the LXRα/nSREBP-1c pathway^{(Reference Ahn, Choi, Hong, Jun, Na, Choi and Kim28)}, which is inhibited by AMPK activation^{(Reference Lee, Hong, Park, Rhee, Park, Oh, Park and Lee34)}. Therefore, the beneficial effect of betaine in hepatic lipogenesis and cholesterol synthesis may be associated with activation of AMPK, which inhibits LXR expression, as well as SREBP-1c and its target genes.

De novo lipogenesis is an integrated process that uses acetyl-CoA to synthesise fatty acids, which are then desaturated and esterified to form triacylglycerol (TG). Liver TG content was associated with VLDL-TG secretion rates, and is exported from hepatocytes in the form of VLDL-TG. Apolipoprotein B100 (apoB100) is lipidated in a process catalyzed by the enzyme microsomal triglyceride transfer protein (MTTP), and is required for VLDL export, suggesting MTTP is important component in maintaining hepatic lipid homeostasis^{(Reference Ipsen, Lykkesfeldt and Tveden-Nyborg35)}. In a recent study, C57BL/6J mice were fed either a high-fat diet or a control diet and received either drinking water treated with 1% betaine or control untreated drinking water 4–6 weeks before timed mating. Throughout gestation, the livers of fetal mice supplemented with betaine had enhanced mRNA expression of MTTP, which promotes VLDL synthesis and secretion, therefore reducing liver triacylglycerol content^{(Reference Joselit, Nanobashvili, Jack-Roberts, Greenwald, Malysheva, Caudill, Saxena and Jiang36)}. Additionally, previous studies have reported that a high-fat diet down-regulates MTTP mRNA expression and that betaine increases lipid transport by microsomal MTTP expression by regulating aberrant DNA methylation^{(Reference Wang, Zhang, Zhou, Liu, Yang, Chen, Zhu, Zheng, Ling and Zhu37)}.

3 Regulation of mitochondrial function

Mitochondria play an important role in regulating hepatic lipid metabolism, and mitochondrial β-oxidation maintains the homeostasis of bioactive lipids^{(Reference Geric, Tyurina, Krysko, Krysko, De Schryver, Kagan, Van Veldhoven, Baes and Verheijden38)}. The mitochondrial respiratory chain is the main subcellular source of reactive oxygen species (ROS), which can damage mitochondrial proteins, lipids and mitochondrial DNA^{(Reference Paradies, Paradies, Ruggiero and Petrosillo39)}. Excessive intake of NEFAs increases bioactive lipid accumulation in mitochondria, leading to the formation of ROS. Increasing evidence has reported that mitophagy or autophagy blockade leads to the accumulation of damaged ROS-generating mitochondria. This in turn leads to mitochondrial dysfunction and endoplasmic reticulum (ER) stress^{(Reference Zhou, Yazdi, Menu and Tschopp40)}, increases mitochondrial biogenesis and inhibits ER stress-mediated ROS, leading to the promotion of cell survival during ER stress in eukaryotic cells^{(Reference Knupp, Arvan and Chang41)}. Moreover, factors such as oxidative stress, alterations in mitochondrial structure and functional mitochondrial dysfunction are particularly susceptible to ROS attack and play an important role in the physiopathology of NAFLD^{(Reference Paradies, Paradies, Ruggiero and Petrosillo39)}. These results suggest that the regulation of mitochondrial function is a fundamental mechanism for hepatoprotection. Interestingly, betaine could play a primary role in the hepatoprotective mechanisms of mitochondria through its antioxidative and mitochondria-regulating properties^{(Reference Adjoumani, Wang, Zhou, Liu and Zhang42,Reference Heidari, Niknahad, Sadeghi, Mohammadi, Ghanbarinejad, Ommati, Hosseini, Azarpira, Khodaei, Farshad, Rashidi, Siavashpour, Najibi, Ahmadi and Jamshidzadeh43)} ; however, the mechanism by which betaine regulates mitochondrial function is still unclear.

Mitochondrial fusion combines two mitochondria into one mitochondrion. Healthy mitochondria eventually fuse to form a healthy mitochondrial pool^{(Reference Mansouri, Gattolliat and Asselah44)}. The reduced signalling for mitochondrial fusion detected in NAFLD animal models underlines its potential role in the pathology of NAFLD^{(Reference Du, Zhang, Han, Man, Zhang, Chu, Nan and Yu45)}. Mitochondrial fission is the division of one mitochondrion into two mitochondria and is mediated by the interaction of cytoplasmic mitochondrial fission-related protein 1 (Drp1) and other mitochondrial fission proteins, such as mitochondrial fission factor (MFF)^{(Reference Hall, Burke, Dongworth and Hausenloy46)}. Moreover, mitochondrial fusion and fission-related mechanisms depend on the available energy^{(Reference Nasrallah and Horvath47)} and the response to metabolic stressors. Ultrastructural mitochondrial lesions, altered mitochondrial dynamics, decreased activity of respiratory chain complexes and impaired ability to synthesise adenosine triphosphate are observed in liver tissues from patients with NAFLD^{(Reference Mansouri, Gattolliat and Asselah44)}. Cells treated with betaine exhibit enhanced mitochondrial and cellular respiration, mitochondrial potential, and ATP production, thereby increasing energy expenditure^{(Reference Lee48)}. A kinase anchoring protein 1 (AKAP1) is a regulator of mitochondrial fusion that is phosphorylated by AMPK, leading to increased fatty acid oxidation^{(Reference Hoffman Nolan, Parker Benjamin, Chaudhuri, Fisher-Wellman Kelsey, Kleinert, Humphrey Sean, Yang, Holliday, Trefely, Fazakerley Daniel, Stöckli, Burchfield James, Jensen Thomas, Jothi, Kiens, Wojtaszewski Jørgen, Richter Erik and James David49)}. AKAP1 plays an important role in anchoring PKA to the cytoplasmic face of the mitochondrial outer membrane^{(Reference Marin50)} and maximises ATP production. In mitochondria, PKA phosphorylates DRP1, leading to mitochondrial fission^{(Reference Hoffman Nolan, Parker Benjamin, Chaudhuri, Fisher-Wellman Kelsey, Kleinert, Humphrey Sean, Yang, Holliday, Trefely, Fazakerley Daniel, Stöckli, Burchfield James, Jensen Thomas, Jothi, Kiens, Wojtaszewski Jørgen, Richter Erik and James David49)}. Moreover, AMPK phosphorylates mitochondrial fission factor (MFF), which is essential for initiating mitochondrial fission,^{(Reference Toyama, Herzig, Courchet, Lewis, Losón, Hellberg, Young, Chen, Polleux, Chan and Shaw51)} and recruits DRP1 to the outer mitochondrial membrane. Therefore, the mechanism by which betaine indirectly regulates mitochondrial function to control the rates of fatty acid oxidation may involve the phosphorylation of AMPK, which regulates mitochondrial fusion and fission. Future studies investigating the mechanism by which betaine increases fatty acid oxidation may involve enhancing mitochondrial fusion and fission through pathways involving AMPK, AKAP1 activity and DRP1 phosphorylation.

Betaine increases mitochondrial membrane potential and cellular respiration, and increases in mitochondrial biogenesis must be balanced by the removal of damaged mitochondria^{(Reference Garcia and Shaw52)}, which is initiated by mitochondrial fission. Mitochondria undergo fusion or fission to maintain mitochondrial energy production and biogenesis to respond to changes in nutrient supplementation^{(Reference Schrepfer and Scorrano53)}. Changes in both the biogenesis and/or mitophagy of mitochondria increase mitochondrial content and cellular energy levels^{(Reference Lee48)}, which adapt to the metabolic needs of cells through a balance between fusion and fission events. A key pathway regulating mitochondrial biogenesis is the master transcription factor and transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) and its target genes nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM). This pathway is required for mitochondrial DNA (mtDNA) transcription and has been linked to metabolic changes leading to increased mitochondrial biogenesis^{(Reference Chen, Tao, Li and Yao54)}. The significant up-regulation of the mtDNA copy number by betaine may be due to up-regulation of TFAM^{(Reference Hu, Sun, Liu, Jia, Cai, Idriss, Omer and Zhao55)}, an activator of mtDNA replication and transcription^{(Reference Picca and Lezza56)}. Additionally, mitochondrial biogenesis provides cellular chemical energy in the form of ATP, which stimulates AMPK activation and further increases GLUT-4 expression and regulation of fatty acid oxidation via ACC phosphorylation^{(Reference Banerjee, Bruckbauer and Zemel57)}. Therefore, betaine increases mitochondrial content to alter mitochondrial biogenesis, possibly via the phosphorylation of ACC and AMPK, which up-regulate the network of transcription factors PGC1α, NRF-1, TFAM and Sirt-1^{(Reference Ma, Meng, Kang, Zhang, Jung and Park26)}. In animal experiments, betaine improves fatty acid oxidation, maintains energy balance and reduces liver fat by up-regulating fibroblast growth factor 21 (FGF21)^{(Reference Ejaz, Martinez-Guino, Goldfine, Ribas-Aulinas, De Nigris, Ribó, Gonzalez-Franquesa, Garcia-Roves, Li, Dreyfuss, Gall, Kim, Bottiglieri, Villarroya, Gerszten, Patti and Lerin18)}, which also significantly increases the number of mitochondria and the expression of mitochondrial biogenesis-related genes, including PGC1α, NRF1 and TFAM^{(Reference Zhang, Li, Wang, Zhan, Wang, Zhong, Guo and Zhang58)}.

Autophagy plays an important role in degrading lipids in damaged cells and can not only regulate lipid metabolism and insulin resistance in mice with diet-induced obesity^{(Reference Li, Gong, Yang, Yang, Fan and Zhou59)} but also protect hepatocytes from injury and cell death^{(Reference Wu, Zhang and Chan60)}. Autophagic flux is impaired in NAFLD patients and NAFLD mouse models, as well as in human hepatocytes with increased ER stress^{(Reference González-Rodríguez, Mayoral, Agra, Valdecantos, Pardo, Miquilena-Colina, Vargas-Castrillón, Lo Iacono, Corazzari, Fimia, Piacentini, Muntané, Boscá, García-Monzón, Martín-Sanz and Valverde61)}. Betaine indirectly phosphorylates unc-51-like autophagy activating kinase 1 (ULK1)^{(Reference Veskovic, Mladenovic, Milenkovic, Tosic, Borozan, Gopcevic, Labudovic-Borovic, Dragutinovic, Vucevic, Jorgacevic, Isakovic, Trajkovic and Radosavljevic13)}, which plays a key role in the initiation stage of autophagy, promotes autophagosome-lysosome fusion^{(Reference Wang, Wang, Zhang, Luo, Liu, Xu, Diao, Liao and Liu62)}, promotes mitophagy^{(Reference Kim, Kundu, Viollet and Guan63)} and responds to mitochondrial fission. Betaine also phosphorylates autophagy activators such as autophagy-related protein 4/5 (ATG4/5), which increases the number of autophagic vesicles and degradation of the autophagic target sequestosome 1/p62 in the livers of NAFLD mice. Betaine also regulates mitophagy through inhibition of mechanistic target of rapamycin complex 1 (mTORC1), which phosphorylates and inhibits ULK1 and its activator Akt^{(Reference Veskovic, Mladenovic, Milenkovic, Tosic, Borozan, Gopcevic, Labudovic-Borovic, Dragutinovic, Vucevic, Jorgacevic, Isakovic, Trajkovic and Radosavljevic13)}. These latter proteins are regulated by AMPK phosphorylation^{(Reference Li, Gong, Yang, Yang, Fan and Zhou59)}. Moreover, AMPK phosphorylation regulates mitochondrial transcription by promoting the import of FOXO3 into mitochondria^{(Reference Celestini and Tezil64)}. FOXO3 promotes the translocation of FOXO1 from the nucleus to the cytoplasm, resulting in an increase in FOXO1-induced autophagy via activation of the AKT1 signalling pathway without increasing the expression of FOXO1 at the protein level^{(Reference Zhou, Liao, Yang, Ma, Li, Wang, Wang, Wang, Zhang, Yin, Zhao and Zhu65)}. Additionally, betaine plays an important role in antiapoptotic pathways and autophagy by increasing the expression of the antiapoptotic protein Bcl-2 and inhibiting the expression of the proapoptotic protein Bax^{(Reference Veskovic, Mladenovic, Milenkovic, Tosic, Borozan, Gopcevic, Labudovic-Borovic, Dragutinovic, Vucevic, Jorgacevic, Isakovic, Trajkovic and Radosavljevic13)}, which impairs mitochondrial membrane integrity. Thus, betaine promotes autophagy by activating AMPK, ULK1, and phosphorylated FOXO1 and FOXO3 and by antagonising mTORC1. However, the molecular mechanism by which betaine ameliorates NAFLD by regulating autophagy in hepatocytes requires further study.

4 Enhancement of fatty acid oxidation through epigenetic function of betaine

Mitochondrial dysfunction increases DNA methylation, which can cause the development of metabolic syndrome in obesity^{(Reference Ejarque, Ceperuelo-Mallafré, Serena, Maymo-Masip, Duran, Díaz-Ramos, Millan-Scheiding, Núñez-Álvarez, Núñez-Roa, Gama, Garcia-Roves, Peinado, Gimble, Zorzano, Vendrell and Fernández-Veledo66)} and the progression of NAFLD^{(Reference Zhou, Yazdi, Menu and Tschopp40,Reference Begriche, Massart, Robin, Bonnet and Fromenty67)} . Therefore, the regulation of DNA methylation could prevent NAFLD by attenuating mitochondrial dysfunction^{(Reference Mishra and Kowluru68)}. DNA cytosine-5-methyltransferases (DNMTs) mediate DNA methylation by catalysing the transfer of the methyl group from SAM to cytosine during DNA replication^{(Reference Yang, Yang, Gong, Rose, Pirgozliev, Chen and Wang69)}. Betaine supplementation significantly elevates DNMT at the mRNA level in animals^{(Reference Idriss, Hu, Sun, Jia, Jia, Omer, Abobaker and Zhao70)}. The epigenetic effects of SAM via DNA methylation are driven by SAM precursors such as betaine and Met. Betaine acts as a methyl donor for DNA methylation in epigenetic regulation and provides a methyl group to participate in one-carbon metabolism to promote SAM formation^{(Reference Deminice, Silva, Lamarre, Kelly, Jacobs, Brosnan and Brosnan25)}. A recent study reported that access to 1% betaine-supplemented water for a total of 8 weeks alters the methylation status of specific gene promoters in mice, leading to persistent changes in gene expression that could be beneficial for the treatment of obesity and type 2 diabetes owing to their valuable metabolic effects on lipolysis, the TCA cycle and mitochondrial oxidative demethylation^{(Reference Sivanesan, Taylor, Zhang and Bakovic15)}. However, the mechanism by which betaine methylation regulates mitochondrial oxidative demethylation to ameliorate NAFLD remains unclear.

Fat mass and obesity-associated (FTO) protein is a demethylase that plays a critical role in demethylation. FTO overexpression enhances lipogenesis and ROS production^{(Reference Bravard, Lefai, Meugnier, Pesenti, Disse, Vouillarmet, Peretti, Rabasa-Lhoret, Laville, Vidal and Rieusset71)} and increases liver damage in NAFLD patients^{(Reference Guo, Ren, Li, Xi, Li, Gao, Liu and Su72)}. Interestingly, FTO-dependent demethylation inhibits the methylation of m6A^{(Reference Wu, Feng, Jiang, Zhou, Jiang, Cai, Wang, Shan and Wang73)}, the most prevalent mRNA modification, thereby reducing lipid accumulation and energy metabolism^{(Reference Zhong, Yu, Frazier, Weng, Li, Cham, Dolan, Zhu, Hubert, Tao, Lin, Martinez-Guryn, Huang, Wang, Liu, He, Chang and Leone74)}. Moreover, FTO-dependent m6A demethylation is associated with AMPK-related signalling pathways, which reduce lipid accumulation in skeletal muscle cells by regulating FTO expression and FTO-dependent m6A demethylation^{(Reference Wu, Feng, Jiang, Zhou, Jiang, Cai, Wang, Shan and Wang73)}. Therefore, AMPK plays an important role in regulating lipid accumulation by inhibiting FTO expression and m6A methylation, providing new insights into the molecular regulation of lipid metabolism. In fact, a high-fat diet induces hepatic steatosis by reducing the expression of lipolysis genes such as hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and these changes are accompanied by increased hepatic FTO and reduced m6A levels. Betaine plays a hepatoprotective role in preventing these changes by rectifying the m6A mRNA hypomethylation state and down-regulating FTO expression in mouse liver^{(Reference Chen, Zhou, Wu, Wang and Wang75)}. Additionally, betaine activates AMPKα1, which is beneficial for m6A methylation, and reduces the expression of FTO in high-fat diet-induced wild-type mice^{(Reference Zhou, Chen, Chen, Wu, Wang and Wang76)}. In adipose tissue, the up-regulation of ATGL and HSL expression significantly increases circulating NEFAs, leading to lipid accumulation in the liver; betaine reverses these changes in gene expression by modifying DNA methylation at the promoter in progeny rats^{(Reference Zhao, Yang, Sun, Feng and Zhao77)}.

Peroxisome proliferator activated receptor α (PPARα), a transcription factor, regulates the transcription of numerous genes encoding enzymes in fatty acid oxidation and transportation^{(Reference Wang, Chen, Tan, Wei, Chang, Jin and Zhu78)} and is involved in the regulation of liver lipid metabolism^{(Reference Ruppert, Park, Xu, Hur, Lee and Kersten79)}. Betaine enhances mitochondrial β-oxidation by decreasing hypermethylation of PPARα^{(Reference Wang, Chen, Tan, Wei, Chang, Jin and Zhu78)} and modifications of CpG methylation at the gene promoter of the mitochondrial fatty acid oxidation-related gene carnitine palmitoyl transferase 1α (CPT1α)^{(Reference Hu, Sun, Liu, Jia, Cai, Idriss, Omer and Zhao55)}, which facilitates fatty acyl-CoA entry into mitochondria. CPT1α is inhibited by ACC production by malonyl-CoA^{(Reference Fentz, Kjøbsted, Birk, Jordy, Jeppesen, Thorsen, Schjerling, Kiens, Jessen, Viollet and Wojtaszewski80-Reference Kim, Lee, Kim, Park, An, Lee, Chung, Park, Yu, Choi and Chung89)} and is regulated by PPARα^{(Reference Xiao, Wang, Yan, Zhou, Cao and Cai81)}. In the context of DNA methylation, Met is metabolised into SAM by methionine adenosyltransferase in the liver, and the synthesis of SAM serves to enhance DNA methylation, leading to greater expression of PPARα and its target gene FGF21 in lipid metabolism^{(Reference Osorio, Jacometo, Zhou, Luchini, Cardoso and Loor82)}. Betaine plays a role in DNA methylation by increasing the levels of BHMT and SAM, which participate in the functions of mitochondrial oxidative enzymes such as PPARα and ACC. These enzymes are localised in the mitochondrial matrix and have valuable metabolic effects on lipolysis, the TCA cycle and mitochondrial oxidative phosphorylation^{(Reference Sivanesan, Taylor, Zhang and Bakovic15)}. Additionally, AMPK increases the expression of PPARα, which in turn enhances the regulatory effect of AMPK on the expression of angiopoietin-like 8 (ANGPTL8), a liver-derived secretory protein that elevates serum triacylglycerol^{(Reference Lee, Hong, Park, Rhee, Park, Oh, Park and Lee34)}.

These studies clarified that the methylation function of betaine plays an essential role in lipid metabolism and links the epigenetic modification of DNA and RNA methylation with lipid accumulation. Future studies should clarify the mechanism by which betaine exerts different regulatory effects on lipid metabolism in different tissues, thereby providing new targets for the regulation of hepatic lipid metabolism and NAFLD.

5 Reduction of insulin resistance

The metabolic processes gluconeogenesis, glycolysis and glycogenolysis coordinately regulate liver glucose, leading to glucose homeostasis. The increase in lipid accumulation induced by a high-fat diet disturbs glucose homeostasis, leading to obesity, which is a major driver of insulin resistance and NAFLD^{(Reference Du, Shen, Tan, Zhang, Zhao, Xu, Gan, Yang, Ma, Jiang, Tang, Jiang, Jin, Li, Bai, Li, Wang, Zhang and Zhu12)}. Interestingly, insulin resistance is closely associated with NAFLD^{(Reference Xia, Chen, Zhu, Huang, Yin and Ren83)}. In fact, betaine supplementation improves insulin sensitivity and reduces insulin resistance to maintain glucose homeostasis^{(Reference Du, Shen, Tan, Zhang, Zhao, Xu, Gan, Yang, Ma, Jiang, Tang, Jiang, Jin, Li, Bai, Li, Wang, Zhang and Zhu12,Reference Ejaz, Martinez-Guino, Goldfine, Ribas-Aulinas, De Nigris, Ribó, Gonzalez-Franquesa, Garcia-Roves, Li, Dreyfuss, Gall, Kim, Bottiglieri, Villarroya, Gerszten, Patti and Lerin18)} ; however, the mechanism by which betaine reduces insulin resistance needs to be elucidated. Recent studies have reported that de novo lipogenesis and oxidative stress are often related to the lack of insulin sensitivity in the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signalling pathway^{(Reference Dekker, Su, Baker, Rutledge and Adeli84,Reference Pan, Guo and Su85)} , which also plays an important role in maintaining glucose homeostasis and alleviating insulin resistance^{(Reference Yan, Dai and Zheng86,Reference Park and Pak87)} . Interestingly, one study reported that the observed beneficial effects of betaine in methionine-choline-deficient diet-induced NAFLD coincide with increased hepatic phosphorylation of Akt^{(Reference Veskovic, Mladenovic, Milenkovic, Tosic, Borozan, Gopcevic, Labudovic-Borovic, Dragutinovic, Vucevic, Jorgacevic, Isakovic, Trajkovic and Radosavljevic13)}. In the insulin/Akt signalling pathway, Akt-dependent phosphorylation inactivates the expression of FOXO1^{(Reference Park and Pak87)}. This inactivation not only controls hepatic glucose production by stimulating the induction of glucose-6-phosphatase (G6PC) and the repression of glucokinase (Gck)^{(Reference Haeusler, Hartil, Vaitheesvaran, Arrieta-Cruz, Knight, Cook, Kammoun, Febbraio, Gutierrez-Juarez, Kurland and Accili88)} but also notably increases lipogenesis and decreases fatty acid oxidation by up-regulating PPARγ and its target genes FAS and ACC expression^{(Reference Kim, Lee, Kim, Park, An, Lee, Chung, Park, Yu, Choi and Chung89)}. It also positively regulates the transcription of CYP7A1, which regulates cholesterol or bile acid metabolism in the liver, thus linking the carbohydrate and cholesterol metabolic pathways^{(Reference Park and Pak87)}. These studies suggest that FOXO1 may induce obesity, insulin resistance and metabolism disorders^{(Reference Guo, Li, You, Li, Hu, Zhang, Miao, Xian, Zhu and Shen90)}. Moreover, AMPK inhibits insulin and AMPK signalling pathways and increases glucose uptake by phosphorylating insulin receptor substrate-1 (IRS), which increases PI3K/AKT phosphorylation^{(Reference Bertrand, Ginion, Beauloye, Hebert, Guigas, Hue and Vanoverschelde91)}. Betaine may phosphorylate IRS-1 to inactivate FOXO1 expression through phosphorylation of PI3K/AKT^{(Reference Xia, Chen, Zhu, Huang, Yin and Ren83)}. Therefore, betaine improves NAFLD by normalising insulin signalling through reduced gluconeogenesis, increased glycogen synthesis and improved hepatic lipid metabolism through the AMPK pathway.

6 Inhibition of liver inflammation by betaine

Lipid metabolism disorders increase the expression of cytochrome P450 2E1 (CYP2E1), which promotes the production of ROS and mitochondrial oxidative stress^{(Reference Zhou, Wan, Shu, Mao, Liu, Yuan, Zhang, Hess, Brüning and Qian92)}. Excessive ROS generation leads to oxidative stress in extrahepatic cells^{(Reference Jin, Ande, Kumar and Kumar93,Reference Zhou, He, Zuo, Zhang, Wan, Long, Huang, Wu, Wu, Liu and Yin94)} . Moreover, excess ROS plays an inhibitory role in AMPK activation^{(Reference Weikel, Ruderman and Cacicedo23)}, and scavenging of ROS by the GSH antioxidant system can prevent AMPK inactivation in mice fed a high-fat diet^{(Reference Zhou, He, Zuo, Zhang, Wan, Long, Huang, Wu, Wu, Liu and Yin94)}. Betaine significantly reduces excess ROS and increases the levels of GSH and glutathione peroxidase to improve the antioxidative stress ability of the liver^{(Reference Veskovic, Mladenovic, Milenkovic, Tosic, Borozan, Gopcevic, Labudovic-Borovic, Dragutinovic, Vucevic, Jorgacevic, Isakovic, Trajkovic and Radosavljevic13)}. In blunt snout bream fed a high-fat diet, 1·2% betaine supplementation significantly improves antioxidant defences by increasing superoxide dismutase, catalase and GSH levels and reverses the increase in malondialdehyde levels^{(Reference Adjoumani, Wang, Zhou, Liu and Zhang42)}. However, betaine supplementation decreases hepatic triacylglycerol accumulation by increasing CYP2E1 expression in ApoE−/− mice^{(Reference Wang, Chen, Tan, Wei, Chang, Jin and Zhu78)}. Therefore, further research is needed to clarify these conflicting effects of betaine treatment.

ROS also induce mitochondrial dysfunction by activating the expression of the nucleotide-binding domain leucine-rich-containing family pyrin domain-containing-3 (NLRP3) inflammasome^{(Reference Zhou, Yazdi, Menu and Tschopp95)}, which is triggered by metabolic dysregulation^{(Reference Du, Zhang, Han, Man, Zhang, Chu, Nan and Yu45)}. Additionally, ROS trigger the inflammatory pathways of nuclear factor kappa B (NF-κB), leading to liver inflammation^{(Reference Cheng, Chen, Liu, Zhao and Cao96)}, which is often mediated by chemokines, including IL1 and IL6. Activation of NF-κB leads to the synthesis of inflammatory mediators, stimulates ROS synthesis, deteriorates oxidative stress^{(Reference Timofte, Toarba, Hogas, Covic, Ciobica, Chirita, Lefter, Arhire, Arcan and Alexinschi97)} and accelerates the development of NAFLD^{(Reference Samuel Varman and Shulman Gerald98,Reference Jian, Ao, Wu, Lv, Ma, Zhao, Tong, Ren, Chen and Li99)} . Therefore, inhibition of ROS production and NF-κB expression significantly reverses hepatic inflammation and NAFLD. Betaine enhances the removal of ROS by depressing the expression of the NLRP3 inflammasome via inactivation of FOXO1^{(Reference Xia, Chen, Zhu, Huang, Yin and Ren83)}. Betaine also inhibits the hepatic NF-κB/NLRP3 inflammasome activation-mediated inflammation signalling pathway in fructose-fed NAFLD rats^{(Reference Ge, Yu, Xu, Li, Fan, Li and Kong100)}. Betaine suppresses NF-κB and IL-1 expression by decreasing the expression of mitogen-activated protein kinases (MAPKs) and IκB/IKK and increases the liver expression of the anti-inflammatory cytokine interleukin-10 (IL10) in methionine-choline-deficient diet-induced NAFLD mice^{(Reference Veskovic, Mladenovic, Milenkovic, Tosic, Borozan, Gopcevic, Labudovic-Borovic, Dragutinovic, Vucevic, Jorgacevic, Isakovic, Trajkovic and Radosavljevic13)}. Additionally, inhibition of the expression of the chemokines IL1, IL6 and IκB through the AMPK/NF-κB signalling pathway is considered a target for inflammatory diseases^{(Reference Ye, Zhu and Sun101)}. Therefore, the anti-inflammatory mechanism of betaine may be mediated by the GSH/AMPK/NF-κB signalling pathways.

7 Regulation of gut dysfunction

The gut microbiome is a functional organ that maintains intestinal homeostasis^{(Reference Zhang, Sun, Zhao, Chen, Fan, Jiao, Zhao, Wang, Li, Li and Lin102)}. There is growing evidence of a close correlation between the gut microbiome and NAFLD^{(Reference He, Ji, Jia and Li104)}. Intestinal injury and increased intestinal permeability accelerate the pathogenesis of NAFLD^{(Reference Jiang, Wu, Wang, Chi, Zhang, Qiu, Hu, Li and Liu105)}. Recent studies have shown that a high-fat diet changes bile acid homeostasis, leading to gut microbiota dysbiosis^{(Reference Yokota, Fukiya, Islam, Ooka, Ogura, Hayashi, Hagio and Ishizuka106-Reference Turnbaugh108)}, which increases hepatic injury, inflammation and NAFLD by influencing lipid metabolism^{(Reference Rastelli, Knauf and Cani109,Reference Cuevas-Sierra, Ramos-Lopez, Riezu-Boj, Milagro and Martinez110)} . Interestingly, bran-enriched diets, which contain abundant betaine, increase the relative abundances of Akkermansia, Bifidobacterium, Coriobacteriaceae, Lactobacillus and Ruminococcus, many of which are beneficial for host health^{(Reference Koistinen, Kärkkäinen, Borewicz, Zarei, Jokkala, Micard, Rosa-Sibakov, Auriola, Aura, Smidt and Hanhineva111)}. Betaine also significantly improves the microbial community in the gut by reducing the abundances of Coriobacteriaceae, Lachnospiraceae, Enterorhabdus and Coriobacteriales and markedly enriching the taxa Bacteroidaceae, Bacteroides, Parabacteroides and Prevotella in a model group^{(Reference Chen, Wang, Jiao, Shi, Pei, Wang and Gong112)}. Moreover, betaine may have beneficial effects via its osmoprotective role^{(Reference Ueland, Holm and Hustad113)} in osmotic regulation and antioxidant activity in human enterocytes. Betaine decreases intestinal injury and intestinal permeability, which limits the entry of bacterial endotoxins into systemic circulation^{(Reference Ommati, Farshad, Mousavi, Jamshidzadeh, Azmoon, Heidari, Azarpira, Niknahad and Heidari114)}. These results suggest that betaine plays a protective role in reducing intestinal cell damage and inflammation to restore gut microbiota homeostasis. However, the mechanism of betaine in the regulation of gut microbiota homeostasis needs to be further studied.

Gut microbial-derived products, many of which are produced by bacterial fermentation, primarily arrive at the liver through the portal circulation^{(Reference Zhu, Sawrey-Kubicek, Bardagjy, Houts, Tang, Sacchi, Randolph, Steinberg and Zivkovic115)}. Lipopolysaccharide (LPS), a component of the Gram-negative bacterial cell membrane and the active component of endotoxin, impairs intestinal permeability^{(Reference Aron-Wisnewsky, Gaborit, Dutour and Clement116)}, and a damaged intestinal mucosal barrier lead to gut dysbiosis. Negative microbial metabolites interact with Toll-like receptor 4 (TLR4)^{(Reference Chen, Wang, Jiao, Shi, Pei, Wang and Gong112)}, a membrane receptor of LPS, triggering an essential inflammatory cascade involving MAPKs and the NF-κB pathway in Kupffer cells^{(Reference Machado and Cortez-Pinto117)}. NF-κB induces the transcription of numerous pro-inflammatory cytokines such as pro-inflammatory tumour necrosis factor-α (TNF-α), which regulates lipid metabolism by impairing insulin signalling by inhibiting IRS-1^{(Reference Alipourfard, Datukishvili and Mikeladze118)}. IL-1β regulates lipid metabolism by suppressing PPARα, resulting in hepatic triacylglycerol accumulation^{(Reference Stienstra, Saudale, Duval, Keshtkar, Groener, van Rooijen, Staels, Kersten and Müller119)}. Additionally, activation of TLR4 triggers the production of pro-inflammatory cytokines, including TNF-α, interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin (IL-12) and interferon gamma (IFN-γ)^{(Reference Jiang, Wu, Wang, Chi, Zhang, Qiu, Hu, Li and Liu105,Reference Abu-Shanab and Quigley120,Reference Alisi, Ceccarelli, Panera and Nobili121)} . Betaine improves intestinal injury to prevent LPS translocation to systemic circulation^{(Reference Ommati, Farshad, Mousavi, Jamshidzadeh, Azmoon, Heidari, Azarpira, Niknahad and Heidari114)} and decreases TLR4 and NF-κB expression and histological scores for steatosis, inflammation and necrosis by inhibiting TLR4 signalling pathways in high-fat diet-induced NAFLD rats^{(Reference Zhang, Wang, Wang, Li, Zhang, Luo, Song and Gong122)}. Betaine effectively improves intestinal injury in acute liver failure mice by inhibiting the TLR4/MyD88 signalling pathway, improving the intestinal mucosal barrier and maintaining the gut microbiota composition^{(Reference Chen, Liu, Zhou, Chen, Wang, Tan, Wang, Zheng, Zhang, Ling and Zhu123)}. The appropriate betaine supplementation level for on-growing grass carp (body weight 210–776 g) is estimated to be 4·28 to 4·51 g/kg diet; betaine boosts the growth performance and enhances the immune function of on-growing grass carp by down-regulating intestinal TNF-α, IL-1β, IFN-γ2, IL-6 and IL-8 mRNA expression partly mediated by the [IKKβ, γ/IκBα/NF-κBp65,c-Rel] signalling pathway. In addition, betaine up-regulates the mRNA expression of the intestinal anti-inflammatory cytokines transforming growth factor TGF-β1 and IL-10, partly mediated by the target of rapamycin (TOR) signalling pathway^{(Reference Sun, Jiang, Wu, Liu, Jiang, Yang, Kuang, Tang, Zhou and Feng124)}. These studies suggest that the inhibition of TLR4/NF-κB signalling pathways warrants further study to elucidate the effect of betaine on gut microbiota modulation.